scholarly journals Genome Evolution and Nitrogen Fixation in Bacterial Ectosymbionts of a Protist Inhabiting Wood-Feeding Cockroaches

2016 ◽  
Vol 82 (15) ◽  
pp. 4682-4695 ◽  
Author(s):  
Vera Tai ◽  
Kevin J. Carpenter ◽  
Peter K. Weber ◽  
Christine A. Nalepa ◽  
Steve J. Perlman ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Genome Evolution ◽  
Outer Membrane Proteins ◽  
Microbial Interactions ◽  
Content Type ◽  
Evolutionary Forces ◽  
Symbiotic Interactions ◽  
Cryptocercus Punctulatus ◽  
Nanosims Analysis

ABSTRACTBy combining genomics and isotope imaging analysis using high-resolution secondary ion mass spectrometry (NanoSIMS), we examined the function and evolution ofBacteroidalesectosymbionts of the protistBarbulanymphafrom the hindguts of the wood-eating cockroachCryptocercus punctulatus. In particular, we investigated the structure of ectosymbiont genomes, which, in contrast to those of endosymbionts, has been little studied to date, and tested the hypothesis that these ectosymbionts fix nitrogen. Unlike with most obligate endosymbionts, genome reduction has not played a major role in the evolution of theBarbulanymphaectosymbionts. Instead, interaction with the external environment has remained important for this symbiont as genes for synthesis of transporters, outer membrane proteins, lipopolysaccharides, and lipoproteins have been retained. The ectosymbiont genome carried two complete operons for nitrogen fixation, a urea transporter, and a urease, indicating the availability of nitrogen as a driving force behind the symbiosis. NanoSIMS analysis ofC. punctulatushindgut symbionts exposedin vivoto15N2supports the hypothesis thatBarbulanymphaectosymbionts are capable of nitrogen fixation. This genomic andin vivofunctional investigation of protist ectosymbionts highlights the diversity of evolutionary forces and trajectories that shape symbiotic interactions.IMPORTANCEThe ecological and evolutionary importance of symbioses is increasingly clear, but the overall diversity of symbiotic interactions remains poorly explored. In this study, we investigated the evolution and nitrogen fixation capabilities of ectosymbionts attached to the protistBarbulanymphafrom the hindgut of the wood-eating cockroachCryptocercus punctulatus. In addressing genome evolution of protist ectosymbionts, our data suggest that the ecological pressures influencing the evolution of extracellular symbionts clearly differ from intracellular symbionts and organelles. Using NanoSIMS analysis, we also obtained direct imaging evidence of a specific hindgut microbe playing a role in nitrogen fixation. These results demonstrate the power of combining NanoSIMS and genomics tools for investigating the biology of uncultivable microbes. This investigation paves the way for a more precise understanding of microbial interactions in the hindguts of wood-eating insects and further exploration of the diversity and ecological significance of symbiosis between microbes.

scholarly journals A Toxic Environment: a Growing Understanding of How Microbial Communities Affect Escherichia coli O157:H7 Shiga Toxin Expression

2020 ◽  
Vol 86 (24) ◽  
Author(s):  
Erin M. Nawrocki ◽  
Hillary M. Mosso ◽  
Edward G. Dudley
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Microbial Interactions ◽  
Severe Illness ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Lambdoid Prophage

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) strains, including E. coli O157:H7, cause severe illness in humans due to the production of Shiga toxin (Stx) and other virulence factors. Because Stx is coregulated with lambdoid prophage induction, its expression is especially susceptible to environmental cues. Infections with Stx-producing E. coli can be difficult to model due to the wide range of disease outcomes: some infections are relatively mild, while others have serious complications. Probiotic organisms, members of the gut microbiome, and organic acids can depress Stx production, in many cases by inhibiting the growth of EHEC strains. On the other hand, the factors currently known to amplify Stx act via their effect on the stx-converting phage. Here, we characterize two interactive mechanisms that increase Stx production by O157:H7 strains: first, direct interactions with phage-susceptible E. coli, and second, indirect amplification by secreted factors. Infection of susceptible strains by the stx-converting phage can expand the Stx-producing population in a human or animal host, and phage infection has been shown to modulate virulence in vitro and in vivo. Acellular factors, particularly colicins and microcins, can kill O157:H7 cells but may also trigger Stx expression in the process. Colicins, microcins, and other bacteriocins have diverse cellular targets, and many such molecules remain uncharacterized. The identification of additional Stx-amplifying microbial interactions will improve our understanding of E. coli O157:H7 infections and help elucidate the intricate regulation of pathogenicity in EHEC strains.

scholarly journals Putative Inv Is Essential for Basolateral Invasion of Caco-2 Cells and Acts Synergistically with OmpA To AffectIn VitroandIn VivoVirulence of Cronobacter sakazakii ATCC 29544

2014 ◽  
Vol 82 (5) ◽  
pp. 1755-1765 ◽  
Author(s):  
Dilini Chandrapala ◽  
Kyumson Kim ◽  
Younho Choi ◽  
Amal Senevirathne ◽  
Dong-Hyun Kang ◽  
...  
Keyword(s):  
Outer Membrane Proteins ◽  
Opportunistic Pathogen ◽  
Additive Effect ◽  
Neonatal Meningitis ◽  
Cronobacter Sakazakii ◽  
Content Type ◽  
Enteric Infections ◽  
Culture Models

ABSTRACTCronobacter sakazakiiis an opportunistic pathogen that causes neonatal meningitis and necrotizing enterocolitis. Its interaction with intestinal epithelium is important in the pathogenesis of enteric infections. In this study, we investigated the involvement of theinvgene in the virulence ofC. sakazakiiATCC 29544in vitroandin vivo. Sequence analysis ofC. sakazakiiATCC 29544invrevealed that it is different from otherC. sakazakiiisolates. In various cell culture models, an Δinvdeletion mutant showed significantly lowered invasion efficiency, which was restored upon genetic complementation. Studying invasion potentials using tight-junction-disrupted Caco-2 cells suggested that theinvgene product mediates basolateral invasion ofC. sakazakiiATCC 29544. In addition, comparison of invasion potentials of double mutant (ΔompA Δinv) and single mutants (ΔompAand Δinv) provided evidence for an additive effect of the two putative outer membrane proteins. Finally, the importance ofinvand the additive effect of putative Inv and OmpA were also proven in anin vivorat pup model. This report is the first to demonstrate two proteins working synergisticallyin vitro, as well asin vivoinC. sakazakiipathogenesis.

scholarly journals Multiple Ehrlichia chaffeensis Genes Critical for Its Persistent Infection in a Vertebrate Host Are Identified by Random Mutagenesis Coupled with In Vivo Infection Assessment

2020 ◽  
Vol 88 (10) ◽  
Author(s):  
Ying Wang ◽  
Arathy D. S. Nair ◽  
Andy Alhassan ◽  
Deborah C. Jaworski ◽  
Huitao Liu ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Fatty Acid Biosynthesis ◽  
Vaccine Development ◽  
Efflux Pump ◽  
Outer Membrane Proteins ◽  
Random Mutagenesis ◽  
Ehrlichia Chaffeensis ◽  
Content Type ◽  
Obligate Intracellular

ABSTRACT Ehrlichia chaffeensis, a tick-transmitted obligate intracellular rickettsial agent, causes human monocytic ehrlichiosis. In recent reports, we described substantial advances in developing random and targeted gene disruption methods to investigate the functions of E. chaffeensis genes. We reported earlier that the Himar1 transposon-based random mutagenesis is a valuable tool in defining E. chaffeensis genes critical for its persistent growth in vivo in reservoir and incidental hosts. The method also aided in extending studies focused on vaccine development and immunity. Here, we describe the generation and mapping of 55 new mutations. To define the critical nature of the bacterial genes, infection experiments were carried out in the canine host with pools of mutant organisms. Infection evaluation in the physiologically relevant host by molecular assays and by xenodiagnoses allowed the identification of many proteins critical for the pathogen’s persistent in vivo growth. Genes encoding proteins involved in biotin biosynthesis, protein synthesis and fatty acid biosynthesis, DNA repair, electron transfer, and a component of a multidrug resistance (MDR) efflux pump were concluded to be essential for the pathogen’s in vivo growth. Three known immunodominant membrane proteins, i.e., two 28-kDa outer membrane proteins (P28/OMP) and a 120-kDa surface protein, were also recognized as necessary for the pathogen’s obligate intracellular life cycle. The discovery of many E. chaffeensis proteins crucial for its continuous in vivo growth will serve as a major resource for investigations aimed at defining pathogenesis and developing novel therapeutics for this and related pathogens of the rickettsial family Anaplasmataceae.

scholarly journals The KbvR Regulator Contributes to Capsule Production, Outer Membrane Protein Biosynthesis, Antiphagocytosis, and Virulence in Klebsiella pneumoniae

2021 ◽  
Vol 89 (5) ◽  
Author(s):  
Li Xu ◽  
Meng Wang ◽  
Jie Yuan ◽  
Hui Wang ◽  
Moran Li ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Outer Membrane ◽  
Capsular Polysaccharide ◽  
Protein Biosynthesis ◽  
Outer Membrane Proteins ◽  
Systemic Infection ◽  
Innate Immune ◽  
Content Type

ABSTRACT Klebsiella pneumoniae is an opportunistic pathogen that mostly affects patients with weakened immune systems, but a few serotypes (especially K1 and K2) are highly invasive and result in systemic infection in healthy persons. The ability to evade and survive the components of the innate immune system is critical in infection. To investigate the role and mechanism of transcription regulator KP1_RS12260 (KbvR) in virulence and defense against the innate immune response, kbvR deletion mutant and complement strains were constructed. The in vivo animal infection assay and in vitro antiphagocytosis assay demonstrate K. pneumoniae KbvR is an important regulator that contributes to virulence and the defense against phagocytosis of macrophages. The transcriptome analysis and phenotype experiments demonstrated that deletion of kbvR decreased production of capsular polysaccharide (CPS) and biosynthesis of partly outer membrane proteins (OMPs). The findings suggest that KbvR is a global regulator that confers pathoadaptive phenotypes, which provide several implications for improving our understanding of the pathogenesis of K. pneumoniae.

scholarly journals Branch Point Identification and Sequence Requirements for Intron Splicing in Plasmodium falciparum

2011 ◽  
Vol 10 (11) ◽  
pp. 1422-1428 ◽  
Author(s):  
Xiaohong Zhang ◽  
Caitlin A. Tolzmann ◽  
Martin Melcher ◽  
Brian J. Haas ◽  
Malcolm J. Gardner ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Branch Point ◽  
Splice Site ◽  
Consensus Sequence ◽  
Nucleophilic Attack ◽  
Luciferase Reporter ◽  
Branch Points ◽  
Content Type ◽  
Evolutionary Forces

ABSTRACTSplicing of mRNA is an ancient and evolutionarily conserved process in eukaryotic organisms, but intron-exon structures vary.Plasmodium falciparumhas an extreme AT nucleotide bias (>80%), providing a unique opportunity to investigate how evolutionary forces have acted on intron structures. In this study, we developed anin vivoluciferase reporter splicing assay and employed it in combination with lariat isolation and sequencing to characterize 5′ and 3′ splicing requirements and experimentally determine the intron branch point inP. falciparum. This analysis indicates thatP. falciparummRNAs have canonical 5′ and 3′ splice sites. However, the 5′ consensus motif is weakly conserved and tolerates nucleotide substitution, including the fifth nucleotide in the intron, which is more typically a G nucleotide in most eukaryotes. In comparison, the 3′ splice site has a strong eukaryotic consensus sequence and adjacent polypyrimidine tract. In four differentP. falciparumpre-mRNAs, multiple branch points per intron were detected, with some at U instead of the typical A residue. A weak branch point consensus was detected among 18 identified branch points. This analysis indicates thatP. falciparumretains many consensus eukaryotic splice site features, despite having an extreme codon bias, and possesses flexibility in branch point nucleophilic attack.

scholarly journals SadA, a Trimeric Autotransporter from Salmonella enterica Serovar Typhimurium, Can Promote Biofilm Formation and Provides Limited Protection against Infection

2011 ◽  
Vol 79 (11) ◽  
pp. 4342-4352 ◽  
Author(s):  
Dhaarini Raghunathan ◽  
Timothy J. Wells ◽  
Faye C. Morris ◽  
Robert K. Shaw ◽  
Saeeda Bobat ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Biofilm Formation ◽  
Protective Antigen ◽  
Outer Membrane Proteins ◽  
Cell Aggregation ◽  
Sub Saharan Africa ◽  
Content Type ◽  
Serovar Typhimurium

ABSTRACTSalmonella entericais a major cause of morbidity worldwide and mortality in children and immunocompromised individuals in sub-Saharan Africa. Outer membrane proteins ofSalmonellaare of significance because they are at the interface between the pathogen and the host, they can contribute to adherence, colonization, and virulence, and they are frequently targets of antibody-mediated immunity. In this study, the properties of SadA, a purported trimeric autotransporter adhesin ofSalmonella entericaserovar Typhimurium, were examined. We demonstrated that SadA is exposed on theSalmonellacell surfacein vitroandin vivoduring infection of mice. Expression of SadA resulted in cell aggregation, biofilm formation, and increased adhesion to human intestinal Caco-2 epithelial cells. Immunization of mice with folded, full-length, purified SadA elicited an IgG response which provided limited protection against bacterial challenge. When anti-SadA IgG titers were enhanced by administering alum-precipitated protein, a modest additional protection was afforded. Therefore, despite SadA having pleiotropic functions, it is not a dominant, protective antigen for antibody-mediated protection againstSalmonella.

scholarly journals Structural Analysis of the Interaction between the Bacterial Cell Division Proteins FtsQ and FtsB

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Danguole Kureisaite-Ciziene ◽  
Aravindan Varadajan ◽  
Stephen H. McLaughlin ◽  
Marjolein Glas ◽  
Alejandro Montón Silva ◽  
...  
Keyword(s):  
Cell Division ◽  
Membrane Proteins ◽  
Bacterial Cell ◽  
Outer Membrane Proteins ◽  
Ring Structure ◽  
Globular Domain ◽  
Bacterial Cell Division ◽  
Content Type ◽  
Division Site

ABSTRACT Most bacteria and archaea use the tubulin homologue FtsZ as its central organizer of cell division. In Gram-negative Escherichia coli bacteria, FtsZ recruits cytosolic, transmembrane, periplasmic, and outer membrane proteins, assembling the divisome that facilitates bacterial cell division. One such divisome component, FtsQ, a bitopic membrane protein with a globular domain in the periplasm, has been shown to interact with many other divisome proteins. Despite its otherwise unknown function, it has been shown to be a major divisome interaction hub. Here, we investigated the interactions of FtsQ with FtsB and FtsL, two small bitopic membrane proteins that act immediately downstream of FtsQ. We show in biochemical assays that the periplasmic domains of E. coli FtsB and FtsL interact with FtsQ, but not with each other. Our crystal structure of FtsB bound to the β domain of FtsQ shows that only residues 64 to 87 of FtsB interact with FtsQ. A synthetic peptide comprising those 24 FtsB residues recapitulates the FtsQ-FtsB interactions. Protein deletions and structure-guided mutant analyses validate the structure. Furthermore, the same structure-guided mutants show cell division defects in vivo that are consistent with our structure of the FtsQ-FtsB complex that shows their interactions as they occur during cell division. Our work provides intricate details of the interactions within the divisome and also provides a tantalizing view of a highly conserved protein interaction in the periplasm of bacteria that is an excellent target for cell division inhibitor searches. IMPORTANCE In most bacteria and archaea, filaments of FtsZ protein organize cell division. FtsZ forms a ring structure at the division site and starts the recruitment of 10 to 20 downstream proteins that together form a multiprotein complex termed the divisome. The divisome is thought to facilitate many of the steps required to make two cells out of one. FtsQ and FtsB are part of the divisome, with FtsQ being a central hub, interacting with most of the other divisome components. Here we show for the first time in detail how FtsQ interacts with its downstream partner FtsB and show that mutations that disturb the interface between the two proteins effectively inhibit cell division.

scholarly journals Peptidoglycan-Associated Cyclic Lipopeptide Disrupts Viral Infectivity

2019 ◽  
Vol 93 (22) ◽  
Author(s):  
Bryan A. Johnson ◽  
Adam Hage ◽  
Birte Kalveram ◽  
Megan Mears ◽  
Jessica A. Plante ◽  
...  
Keyword(s):  
Viral Infections ◽  
Bacterial Species ◽  
Enteric Viruses ◽  
Hemorrhagic Fever ◽  
Inhibitory Effect ◽  
Microbial Interactions ◽  
Enveloped Viruses ◽  
Content Type ◽  
Cyclic Lipopeptide

ABSTRACT Enteric viruses exploit bacterial components, including lipopolysaccharides (LPS) and peptidoglycan (PG), to facilitate infection in humans. Because of their origin in the bat enteric system, we wondered if severe acute respiratory syndrome coronavirus (SARS-CoV) or Middle East respiratory syndrome CoV (MERS-CoV) also use bacterial components to modulate infectivity. To test this question, we incubated CoVs with LPS and PG and evaluated infectivity, finding no change following LPS treatment. However, PG from Bacillus subtilis reduced infection >10,000-fold, while PG from other bacterial species failed to recapitulate this. Treatment with an alcohol solvent transferred inhibitory activity to the wash, and mass spectrometry revealed surfactin, a cyclic lipopeptide antibiotic, as the inhibitory compound. This antibiotic had robust dose- and temperature-dependent inhibition of CoV infectivity. Mechanistic studies indicated that surfactin disrupts CoV virion integrity, and surfactin treatment of the virus inoculum ablated infection in vivo. Finally, similar cyclic lipopeptides had no effect on CoV infectivity, and the inhibitory effect of surfactin extended broadly to enveloped viruses, including influenza, Ebola, Zika, Nipah, chikungunya, Una, Mayaro, Dugbe, and Crimean-Congo hemorrhagic fever viruses. Overall, our results indicate that peptidoglycan-associated surfactin has broad viricidal activity and suggest that bacteria by-products may negatively modulate virus infection. IMPORTANCE In this article, we consider a role for bacteria in shaping coronavirus infection. Taking cues from studies of enteric viruses, we initially investigated how bacterial surface components might improve CoV infection. Instead, we found that peptidoglycan-associated surfactin is a potent viricidal compound that disrupts virion integrity with broad activity against enveloped viruses. Our results indicate that interactions with commensal bacterial may improve or disrupt viral infections, highlighting the importance of understanding these microbial interactions and their implications for viral pathogenesis and treatment.

scholarly journals MetR-Regulated Vibrio cholerae Metabolism Is Required for Virulence

mBio ◽  
2012 ◽  
Vol 3 (5) ◽  
Author(s):  
Ryan W. Bogard ◽  
Bryan W. Davies ◽  
John J. Mekalanos
Keyword(s):  
Amino Acid ◽  
Vibrio Cholerae ◽  
Virulence Factor ◽  
Current Knowledge ◽  
Intestinal Colonization ◽  
Wild Type ◽  
Pathogenic Potential ◽  
Content Type ◽  
Mouse Intestine

ABSTRACTLysR-type transcriptional regulators (LTTRs) are the largest, most diverse family of prokaryotic transcription factors, with regulatory roles spanning metabolism, cell growth and division, and pathogenesis. Using a sequence-defined transposon mutant library, we screened a panel ofV. choleraeEl Tor mutants to identify LTTRs required for host intestinal colonization. Surprisingly, out of 38 LTTRs, only one severely affected intestinal colonization in the suckling mouse model of cholera: the methionine metabolism regulator, MetR. Genetic analysis of genes influenced by MetR revealed thatglyA1andmetJwere also required for intestinal colonization. Chromatin immunoprecipitation of MetR and quantitative reverse transcription-PCR (qRT-PCR) confirmed interaction with and regulation ofglyA1, indicating that misregulation ofglyA1is likely responsible for the colonization defect observed in themetRmutant. TheglyA1mutant was auxotrophic for glycine but exhibited wild-type trimethoprim sensitivity, making folate deficiency an unlikely cause of its colonization defect. MetJ regulatory mutants are not auxotrophic but are likely altered in the regulation of amino acid-biosynthetic pathways, including those for methionine, glycine, and serine, and this misregulation likely explains its colonization defect. However, mutants defective in methionine, serine, and cysteine biosynthesis exhibited wild-type virulence, suggesting that these amino acids can be scavenged in vivo. Taken together, our results suggest that glycine biosynthesis may be required to alleviate an in vivo nutritional restriction in the mouse intestine; however, additional roles for glycine may exist. Irrespective of the precise nature of this requirement, this study illustrates the importance of pathogen metabolism, and the regulation thereof, as a virulence factor.IMPORTANCEVibrio choleraecontinues to be a severe cause of morbidity and mortality in developing countries. Identification ofV. choleraefactors critical to disease progression offers the potential to develop or improve upon therapeutics and prevention strategies. To increase the efficiency of virulence factor discovery, we employed a regulator-centric approach to multiplex our in vivo screening capabilities and allow whole regulons inV. choleraeto be interrogated for pathogenic potential. We identified MetR as a new virulence regulator and serine hydroxymethyltransferase GlyA1 as a new MetR-regulated virulence factor, both required byV. choleraeto colonize the infant mouse intestine. Bacterial metabolism is a prerequisite to virulence, and current knowledge of in vivo metabolism of pathogens is limited. Here, we expand the known role of amino acid metabolism and regulation in virulence and offer new insights into the in vivo metabolic requirements ofV. choleraewithin the mouse intestine.

scholarly journals Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage

2015 ◽  
Vol 197 (17) ◽  
pp. 2792-2809 ◽  
Author(s):  
Sarita Mallik ◽  
Ellen M. Popodi ◽  
Andrew J. Hanson ◽  
Patricia L. Foster
Keyword(s):  
Dna Damage ◽  
Dna Polymerase ◽  
Dna Helicase ◽  
Dna Lesions ◽  
Replication Forks ◽  
Content Type ◽  
Pol Iv ◽  
Dna Polymerase Iv ◽  
Stalled Replication Forks

ABSTRACTEscherichia coli's DNA polymerase IV (Pol IV/DinB), a member of the Y family of error-prone polymerases, is induced during the SOS response to DNA damage and is responsible for translesion bypass and adaptive (stress-induced) mutation. In this study, the localization of Pol IV after DNA damage was followed using fluorescent fusions. After exposure ofE. colito DNA-damaging agents, fluorescently tagged Pol IV localized to the nucleoid as foci. Stepwise photobleaching indicated ∼60% of the foci consisted of three Pol IV molecules, while ∼40% consisted of six Pol IV molecules. Fluorescently tagged Rep, a replication accessory DNA helicase, was recruited to the Pol IV foci after DNA damage, suggesting that thein vitrointeraction between Rep and Pol IV reported previously also occursin vivo. Fluorescently tagged RecA also formed foci after DNA damage, and Pol IV localized to them. To investigate if Pol IV localizes to double-strand breaks (DSBs), an I-SceI endonuclease-mediated DSB was introduced close to a fluorescently labeled LacO array on the chromosome. After DSB induction, Pol IV localized to the DSB site in ∼70% of SOS-induced cells. RecA also formed foci at the DSB sites, and Pol IV localized to the RecA foci. These results suggest that Pol IV interacts with RecAin vivoand is recruited to sites of DSBs to aid in the restoration of DNA replication.IMPORTANCEDNA polymerase IV (Pol IV/DinB) is an error-prone DNA polymerase capable of bypassing DNA lesions and aiding in the restart of stalled replication forks. In this work, we demonstratein vivolocalization of fluorescently tagged Pol IV to the nucleoid after DNA damage and to DNA double-strand breaks. We show colocalization of Pol IV with two proteins: Rep DNA helicase, which participates in replication, and RecA, which catalyzes recombinational repair of stalled replication forks. Time course experiments suggest that Pol IV recruits Rep and that RecA recruits Pol IV. These findings providein vivoevidence that Pol IV aids in maintaining genomic stability not only by bypassing DNA lesions but also by participating in the restoration of stalled replication forks.

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